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WO1992000983A1 - Composition non corrosive et procedes utiles dans l'extraction d'acides nucleiques - Google Patents

Composition non corrosive et procedes utiles dans l'extraction d'acides nucleiques Download PDF

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Publication number
WO1992000983A1
WO1992000983A1 PCT/US1991/004932 US9104932W WO9200983A1 WO 1992000983 A1 WO1992000983 A1 WO 1992000983A1 US 9104932 W US9104932 W US 9104932W WO 9200983 A1 WO9200983 A1 WO 9200983A1
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WO
WIPO (PCT)
Prior art keywords
extraction
benzyl alcohol
group
nucleic acid
sample
Prior art date
Application number
PCT/US1991/004932
Other languages
English (en)
Inventor
Jeffrey Van Ness
B. Melina Cimler
Rich B. Meyer, Jr.
Nicholaas Marthinus Johannes Vermeulen
Original Assignee
Microprobe Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US07/649,389 external-priority patent/US5130423A/en
Application filed by Microprobe Corporation filed Critical Microprobe Corporation
Priority to DE69130394T priority Critical patent/DE69130394T2/de
Priority to CA002087105A priority patent/CA2087105C/fr
Priority to EP91914813A priority patent/EP0539515B1/fr
Priority to JP03513632A priority patent/JP3102569B2/ja
Publication of WO1992000983A1 publication Critical patent/WO1992000983A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • Organic solvents such as phenol and chloroform are traditionally used in techniques employed to isolate nucleic acid from procary ⁇ tic and eucaryotic cells or from complex biological samples. Nucleic acid isolations typically begin with an enzymatic digest performed with proteases followed by cell lysis using ionic detergents, and then extraction with phenol or a phenol/chloroform combination. The organic and aqueous phases are separated and nucleic acid which has partitioned into the aqueous phase is recovered by precipitation with alcohol.
  • phenol or a phenol/chloroform mixture is corrosive to human skin and is considered as hazardous waste, which must be carefully handled and properly discarded. Further, the extraction method is time consuming and laborious. Marmur, J. Mol . Biol .
  • chaotropic agents such as guanidinium thiocyanate (GnSCN) are widely used to lyse and release nucleic acid from cells into solution, largely due to the fact that chaotropic salts inhibit nucleases and proteases.
  • GnSCN guanidinium thiocyanate
  • nucleic acids all of the above traditional nucleic acid extraction procedures, thus, require the use of toxic compounds such as phenol and/or chloroform.
  • researchers have long sought safer effective extraction procedures. for the extraction of nucleic acids.
  • methods are described for isolating nucleic acid from a sample containing a mixture of nucleic acids and other biological compounds, wherein the sample is combined with an extraction solution containing at least one organic compound, such as benzyl alcohol or a benzyl alcohol derivative, forming an aqueous and non-aqueous phase.
  • the nucleic acid is isolated from the aqueous phase.
  • the resulting combined solution also contains bentonite or Macaloid, as defined below.
  • the sample will first be combined with a lysing agent before extraction.
  • the lysing agents preferred are chaotropic salts, such as guanidinium hydrochloride (GuHCl) and guanidinium isothiocyanate (GuSCN) .
  • FIGURES Figure 1 shows the electrophoretic profile of total nucleic acids (chromosomal DNA and RNA) extracted using as the organic phase: 2-methyl-benzyl alcohol in lane 1; 4-methoxy- benzyl alcohol in lane 2; 3-ethoxy-benzyl alcohol in lane 3; 4-phenoxy-benzyl alcohol in lane 4; benzyl-alcohol in lane 5; and phenol in lane 6.
  • Figure 2 shows the electrophoretic profile of total nucleic acids from extracted fecal samples using benzyl alcohol and bentonite.
  • lane 1 is shown the electrophoretic profile of total nucleic acids isolated from fecal samples spiked with bacteria (Bacteroides gingivalis) when the extraction solution is composed of benzyl alcohol and 1% W/V bentonite.
  • lane 2 shows the profile when the extraction solution is composed of phenol. In both extractions, the recovery of total nucleic acids is comparable, and 16S and 23S rRNAs are isolated in the intact form.
  • the extraction methods of the present invention may be applied to a sample containing a biological mixture of nucleic acids (ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) ) and other biological compounds, which are not nucleic acids.
  • a sample may include any aqueous mixture of biological material which contains nucleic acids, including complex biological mixtures of any eucaryotic and/or procaryotic cells, including protoplasts, or other biological materials which may harbor nucleic acids.
  • tissue culture animal cells animal tissue (e.g., heart, liver or brain, homogenized in lysis buffer) , feces, blood cells, reticulocytes, lymphocytes, plant cells or other cells sensitive to osmotic shock, and cells of bacteria, yeasts, viruses, mycoplasmas, protozoa, rickettsia, fungi and other small microbial cells and the like.
  • the nucleic acid sample to be assayed or to serve as a source for the extraction of nucleic acids is combined with an extraction solution which allows for the separation of the nucleic acids in the sample from the other biological components resident in the sample.
  • the extraction solutions comprise benzyl alcohol, derivatives of benzyl alcohol or other solvents with similar properties.
  • the solvent must effectively cause the nucleic acids to be isolated from other biological components by partitioning the nucleic acids into an aqueous phase upon mixing the extraction solution with, an aqueous sample.
  • the extraction solution will typically comprise an organic composition having an organic compound with the following properties:
  • R 2 , R 3 and R* must be -H;
  • R 2 is -C 6 H 5 , -0CH 2 C 6 H 5 , or -CH 2 C 6 H 5 ,
  • R 3 must be -CH 3 , -OH, -Cl, -Br, -F, -I,
  • R 1 is -(CH 2 ) n OH and n is 2 to 6, then R 3 and R* must both be -H.
  • Formula I is intended to include all iso er forms of the alkyls, both branched and unbranched. Unless otherwise stated, all number ranges are inclusive of the stated range, with the use of zero referring to the absence of the element. For example, -(CH 2 ) n CH 3 , in which n is 0 to 2, refers to the three components: -CH 3 , -CH 2 CH 3 and -CH 2 CH 2 CH 3 .
  • HC 6 H 5 is a benzene ring.
  • R 3 and R* are both -H.
  • the bentonite is first water-saturated and added to the extraction solution.
  • Macaloid® a clay
  • Bentone® are available from N.L. Chemicals, Hightstown, * N. . Bentone® is particularly suitable for use within the present invention because the particulate matter remains in relatively homogeneous suspension for extended periods of time, as compared to bentonite and Macaloid®. It may be necessary to first purify the nuclease inhibitor, for instance bentonite or Macaloid®, as described in Sambrook et al . , Molecular Cloning - A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) , which is incorporated by reference herein. For efficient extraction, it is preferred that small uniform particles be used.
  • the inclusion of dye in the extraction solution results in a colored organic phase and a colorless aqueous phase, thereby provided enhanced delineation of the two phases during the extraction process.
  • the dye concentration is sufficient to provide visual contrast between the aqueous and organic phases, and typically is in the range of about 0.0005- 0.01% (w/v) . Because the dye is resident in the organic phase, the preferred organic compound may vary with choice of dye.
  • the biological samples are typically lysed through the use of lysing agents, prior to or during extraction, to disturb the protein structure and to release the nucleic acid. Preferably, the sample will be subjected to lysis prior to the addition of the extraction solution. Any lysing agents typically used in nucleic acid extraction procedures are appropriate, such as SDS, lysozyme, Proteinase K in various combinations with chaotropic agents, or any other agent which would weaken or disrupt the integrity of the cell membrane.
  • Chaotropic agents which disturb the secondary and tertiary structure of proteins (for example, guanidinium salts such as guanidinium hydr ⁇ chloride (GuHCl) , guanidinium isothio ⁇ yanate (GuSCN) , urea or other isothiocyanates) , may be used as lysing agents, in combination with or prior to the extraction solution, to dissociate nucleic acids and inhibit nucleases.
  • guanidinium salts such as guanidinium hydr ⁇ chloride (GuHCl) , guanidinium isothio ⁇ yanate (GuSCN) , urea or other isothiocyanates
  • chaotropic agents in the extraction and hybridization of nucleic acids is described in E.P Publication No. 0 127 327, which is incorporated by reference herein.
  • labels include ligands which bind to labelled antibodies, fluorophores, che iluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labelled ligand.
  • the choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.
  • nucleic acid Precipitation of the nucleic acid was allowed to occur for 5 minutes at 19°C. The nucleic acids were then pelleted from
  • Fecal samples in transport medium (Trend Fekal** Enteric Plus Transport System, Trend Scientific Inc., St. Paul, Minnesota) were aliquoted into 100 ⁇ l portions. Each aliquot was lysed with 300 ⁇ l of GuSCN lysis solution, as in Example 1, and were spiked with 5 x 10 8 Bacteriodes gingivalis cells. Following spiking, the samples were divided into two equal sets.
  • the solid supports were then washed 5 times with APB, TMNZ (0.05 M Tris (pH 9.5), 1 mM MgCl 2 , 0.5 mM ZnCl 2 ) , and then the presence of alkaline phosphatase was determined by incubating the nylon beads with 150 ⁇ l of 0.5 mM 4-methyl-umbelliferyl phosphate (4-hydroxy- methyl coumarin) in black microtiter well strips (Dynatek Laboratories, Chantilly, VA) . Incubation was for 30 minutes at 37°C. The solution was then decanted and placed in a 96 well microtiter plate. The plates were then directly read using a Fluoroskan II fluorometer (Flow Laboratories, McLean, VA) using an excitation wavelength of 360 nm and an emission wavelength of 456 nm. The results are shown in Table 1 below.
  • DNA extracted according to the protocol set forth in this Example is suitable for use in target amplification procedures, such as PCR (polymerase chain reaction) .
  • target amplification procedures such as PCR (polymerase chain reaction) .
  • PCR polymerase chain reaction
  • Table 3 compares extraction of DNA from whole blood using a Proteinase K/phenol extraction method versus the extraction method of this Example. Briefly, three PCR-positive HTLV-I blood samples and five control blood samples (HTLV-I negative by PCR) were collected in tubes containing sodium EDTA. Each of the positive samples was serially diluted in 5- fold steps with the control blood. Duplicate 100 ⁇ l blood samples were extracted using a Proteinase K/phenol procedure (see, for example, Sambrook et al . , in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory Press, New or , ; . guc , n mp ca ons.
  • the preparation was centrifuged at 12,000 x g for 5 minutes. The supernatant was discarded, the pellet was air dried, and the nucleic acid pellet was then resuspended in RNase-free water or appropriate buffer to a desired concentration.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Physics & Mathematics (AREA)
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  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
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  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Procédés sans danger et efficaces d'extraction d'acides nucléiques. L'invention concerne notamment des procédés d'isolation d'acide nucléique à partir d'un échantillon contenant un mélange biologique d'acides nucléiques et d'autres composés biologiques, dans lesquels l'échantillon est combiné avec une solution d'extraction contenant au moins un composé organique tel que de l'alcool benzoïque ou un dérivé d'alcool benzoïque afin de former une phase aqueuse et une phase non aqueuse. On isole l'acide nucléique de la phase aqueuse. La solution combinée obtenue contient également de préférence une bentonite ayant la notation ci-définie. Typiquement, on combine premièrement l'échantillon avec un agent lytique avant l'extraction. Les agents lytiques préférés sont des sels chaotropes tels que l'hydrochlorure de guanidinium et l'isothiocyanate de guanidinium.
PCT/US1991/004932 1990-07-13 1991-07-12 Composition non corrosive et procedes utiles dans l'extraction d'acides nucleiques WO1992000983A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
DE69130394T DE69130394T2 (de) 1990-07-13 1991-07-12 Nicht-ätzende zusammensetzung und verfahren zur gewinnung von nukleinsäuren
CA002087105A CA2087105C (fr) 1990-07-13 1991-07-12 Composition non corrosive et methodes utilisees pour extraire les acides nucleiques
EP91914813A EP0539515B1 (fr) 1990-07-13 1991-07-12 Composition non corrosive et procedes utiles dans l'extraction d'acides nucleiques
JP03513632A JP3102569B2 (ja) 1990-07-13 1991-07-12 核酸の抽出に有用な非腐食性組成物および方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US55274590A 1990-07-13 1990-07-13
US552,745 1990-07-13
US649,389 1991-02-01
US07/649,389 US5130423A (en) 1990-07-13 1991-02-01 Non-corrosive compositions and methods useful for the extraction of nucleic acids

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WO1992000983A1 true WO1992000983A1 (fr) 1992-01-23

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EP (1) EP0539515B1 (fr)
JP (1) JP3102569B2 (fr)
AT (1) ATE172466T1 (fr)
CA (1) CA2087105C (fr)
DE (1) DE69130394T2 (fr)
ES (1) ES2125869T3 (fr)
WO (1) WO1992000983A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0554034A1 (fr) * 1992-01-28 1993-08-04 Piotr Chomczynski Produit planche-stable et procédé pour isoler RNA, DNA et proteines
WO1994016103A3 (fr) * 1993-01-08 1994-09-01 Hybridon Inc Detection d'oligonucleotides synthetiques extraits de liquides ou de tissus organiques
WO1996000228A1 (fr) * 1994-06-23 1996-01-04 Dade International Inc. Procede d'isolation rapide d'acide nucleique
US5558992A (en) * 1993-01-08 1996-09-24 Hybridon, Inc. Detection of synthetic oligonucleotides extracted from body fluids or tissues
WO1997005248A3 (fr) * 1995-07-31 1997-03-06 Piotr Chomczynski Solution contenant un agent chaotrope et son procede d'utilisation pour l'isolation d'adn, d'arn et de proteines
EP0792932A4 (fr) * 1995-08-21 1999-10-20 Sanko Junyaku Kk Co-precipitant et procede d'extraction d'acides nucleiques
EP0853680A4 (fr) * 1995-10-02 2000-09-13 Novagen Inc Procede permettant de precipiter un acide nucleique a l'aide d'un porteur visible
WO2002014548A1 (fr) * 2000-08-10 2002-02-21 Applied Gene Technologies, Inc. Compositions et procedes de traitement et d'amplification d'echantillons d'acides nucleiques
US6448047B2 (en) 1999-07-30 2002-09-10 Applied Gene Technologies, Inc. Sample processing to release nucleic acids for direct detection
WO2003097831A1 (fr) * 2002-05-17 2003-11-27 Gl Bio Tech Gmbh Procede d'extraction et de purification d'acides nucleiques
WO2004108925A1 (fr) * 2003-06-04 2004-12-16 Qiagen As Methode d'isolation sequentielle d'adn et d'arn a partir du meme echantillon contenant des acides nucleiques
WO2005083122A3 (fr) * 2004-02-27 2006-03-23 Id Voor Groningen En Drenthe S Methode de detection de micro-organisme dans un echantillon fecal

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2153215A1 (fr) * 1994-07-06 1996-01-07 Lu Wang Traitement de tissus enchasses dans la paraffine pour l'analyse genique
DE10222133A1 (de) * 2002-05-17 2003-12-04 Gl Biotech Gmbh Verfahren zur Nukleinsäure-Extraktion und Nukleinsäure-Reinigung

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4440778A (en) * 1981-02-26 1984-04-03 Kowa Co., Ltd. Anti-inflammatory analgesic cataplasm and process for producing the same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4440778A (en) * 1981-02-26 1984-04-03 Kowa Co., Ltd. Anti-inflammatory analgesic cataplasm and process for producing the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Biochemical Journal, Volume 64, issued 1956, KIRBY, "A New Method for the Isolation of Ribonucleic Acids from Mammalian Tissues", pages 405-408, see entire document. *
Biochemical Journal, Volume 66, issued 1957, KIRBY, "A New Method for the Isolation of Deoxyribonucleic Acids: Evidence on the Nature of Bonds between Deoxyribonucleic Acid and Protein", pages 495-504, see entire document. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0554034A1 (fr) * 1992-01-28 1993-08-04 Piotr Chomczynski Produit planche-stable et procédé pour isoler RNA, DNA et proteines
WO1994016103A3 (fr) * 1993-01-08 1994-09-01 Hybridon Inc Detection d'oligonucleotides synthetiques extraits de liquides ou de tissus organiques
US5558992A (en) * 1993-01-08 1996-09-24 Hybridon, Inc. Detection of synthetic oligonucleotides extracted from body fluids or tissues
AU678821B2 (en) * 1993-01-08 1997-06-12 Hybridon, Inc. Detection of synthetic oligonucleotides extracted from body fluids or tissues
US5693466A (en) * 1993-01-08 1997-12-02 Hybridon, Inc. Detection of synthetic oligonucleotides extracted from body fluids or tissues
WO1996000228A1 (fr) * 1994-06-23 1996-01-04 Dade International Inc. Procede d'isolation rapide d'acide nucleique
WO1997005248A3 (fr) * 1995-07-31 1997-03-06 Piotr Chomczynski Solution contenant un agent chaotrope et son procede d'utilisation pour l'isolation d'adn, d'arn et de proteines
US5945515A (en) * 1995-07-31 1999-08-31 Chomczynski; Piotr Product and process for isolating DNA, RNA and proteins
EP0792932A4 (fr) * 1995-08-21 1999-10-20 Sanko Junyaku Kk Co-precipitant et procede d'extraction d'acides nucleiques
US6815541B1 (en) 1995-08-21 2004-11-09 Palma Bee'z Research Institute Co., Ltd. Coprecipitant and method for extracting nucleic acids
EP0853680A4 (fr) * 1995-10-02 2000-09-13 Novagen Inc Procede permettant de precipiter un acide nucleique a l'aide d'un porteur visible
US6448047B2 (en) 1999-07-30 2002-09-10 Applied Gene Technologies, Inc. Sample processing to release nucleic acids for direct detection
WO2002014548A1 (fr) * 2000-08-10 2002-02-21 Applied Gene Technologies, Inc. Compositions et procedes de traitement et d'amplification d'echantillons d'acides nucleiques
WO2003097831A1 (fr) * 2002-05-17 2003-11-27 Gl Bio Tech Gmbh Procede d'extraction et de purification d'acides nucleiques
WO2004108925A1 (fr) * 2003-06-04 2004-12-16 Qiagen As Methode d'isolation sequentielle d'adn et d'arn a partir du meme echantillon contenant des acides nucleiques
WO2005083122A3 (fr) * 2004-02-27 2006-03-23 Id Voor Groningen En Drenthe S Methode de detection de micro-organisme dans un echantillon fecal

Also Published As

Publication number Publication date
CA2087105C (fr) 2000-09-12
DE69130394T2 (de) 1999-04-08
EP0539515B1 (fr) 1998-10-21
EP0539515A4 (fr) 1995-04-26
JPH05508658A (ja) 1993-12-02
JP3102569B2 (ja) 2000-10-23
ES2125869T3 (es) 1999-03-16
ATE172466T1 (de) 1998-11-15
DE69130394D1 (de) 1998-11-26
EP0539515A1 (fr) 1993-05-05
CA2087105A1 (fr) 1992-01-14

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